The present invention relates generally to the field of plant molecular biology, and more particularly to novel polynucleotide sequences and proteins encoded from such sequences derived from Bacillus thuringiensis and that encode ET29, TIC809, ET37, TIC810 and TIC812 proteins that exhibit toxicity to coleopteran species and to insects within the super-order referred to as Hemiptera. Coleopteran toxic proteins include ET29, TIC809 (an amino acid sequence variant of ET29), and ET37 (a homologue of ET29). TIC810 and TIC812 and nucleotide sequences encoding these proteins are also provided herein. When TIC810 or TIC812 are combined together with ET29, TIC809 or ET37, insecticidal compositions are provided that exhibit surprisingly greater potency against coleopteran species as compared to the presentation of only ET29, TIC809, or ET37 alone, and the combination of the two (TIC810 with either ET29, TIC809, or ET37, or TIC812 with either ET29, TIC809, or ET37) surprisingly provides a Hemiptera toxic composition, particularly when provided in the diet of species such as Lygus hesperus (western tarnished plant bug, WTPB). Methods of making and using polynucleotides encoding these and related proteins in the development of transgenic plants and plant cells that are resistant to Coleoptera and Hemiptera insect infestation are also disclosed.
Environmentally-sensitive methods and compositions for controlling or eradicating insect infestation are desirable in many instances because crops of commercial interest are often the targets of insect attack, particularly attack from coleopteran and lepidopteran insect pests. This is particularly true for farmers, nurserymen, growers, and commercial and residential areas which seek to control insect populations using environmentally friendly methods and compositions. Controlling or eradicating Hemiptera infestations of crops is also of commercial importance, and is increasing in importance as biotechnology approaches for coleopteran and Lepidopteran pest control methods become more widely available, particularly because fewer chemical insecticidal applications are utilized, which result in broad spectrum insecticidal activity.
The insecticidal properties of the bacterium Bacillus thuringiensis have been long recognized. B. thuringiensis is well known for producing proteinaceous parasporal crystals, or δ-endotoxins, that exhibit specific toxicity to a variety of lepidopteran, coleopteran, and dipteran larvae (English et. al., U.S. Pat. No. 6,063,597). Compositions comprising B. thuringiensis strains that produce insecticidal proteins have been used commercially as environmentally acceptable insecticides because they exhibit toxicity to specific target insects, and fail to exhibit toxicity to plants, animals and other non-target organisms.
More than 250 different δ-endotoxins have been isolated and characterized. Sequences encoding some of these δ-endotoxins have been used to construct genetically engineered B. thuringiensis products in which one or more insecticidal proteins are expressed that exhibit specific insecticidal activity to target pests, and have been approved for agricultural use as topically applied insecticidal compositions. Transgenic plants expressing one or more Bt insecticidal delta endotoxin proteins for use in controlling one or more insects within a specific class, such as Lepidopteran or coleopteran pests, have been approved for commercialization and have been successful. However, there is a risk that populations of target pest insects that feed on these transgenic plants will develop resistance to one or more of the toxins produced by the plants, and so there remains a need for identifying new insecticidal proteins that can be used alone or together with others that manifest their toxic effects through different modes of action. New insecticidal compositions are desirable for producing transgenic plants that express one or more B. thuringiensis insecticidal proteins toxic to the same insect species, providing a means for managing resistance and delaying or eliminating the development of resistance of any particular susceptible insect species to any of the one or more insecticidal agents expressed within a transgenic plant.
Most Bt toxins exhibit toxicity to lepidopteran species. Few have been shown to be effective against coleopteran species, and other than cytolytic toxins which exhibit no host range specificity, no Bt toxins have been shown to exhibit insecticidal activity to lepidopteran or coleopteran species and to Hemipteran species of insect pests. Thus there is a need for identifying new coleopteran and/or Hemipteran specific insecticidal compositions, and methods for controlling infestations by members of the Coleoptera and Hemiptera insect families, particularly for Coleoptera, by members of the family Chrysomelidae, more particularly, by the genus Diabrotica in the family Chrysomelidae that may include those that are from the genus Diabrotica including Diabrotica virgifera (western corn rootworm, WCR), Diabrotica undecempunctata (southern corn rootworm, SCR), Diabrotica barberi (Northern Corn Rootworm, NCR), Diabrotica virgifera zeae (Mexican Corn Rootworm, MCR), Diabrotica balteata (Brazilian Corn Rootworm, BZR) and Brazilian Corn Rootworm complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa), and particularly by members of the super-order Hemiptera, which includes any insect pest within the sub-order Heteroptera, including insects commonly referred to as stink bugs, Lygus bugs (including Lygus Hesperus, Lygus lineolorus, and Lygus elisus), assassin bugs, bed bugs, and flower bugs, and the sub-order Homoptera, including insects commonly known as cicadas, aphids, leafhoppers, scale insects and whiteflies.